Figure 1: Treatment of Jurkat cells with TSA causes accumulation of acetylation on Histone H4, detected by Sandwich ELISA Kit #7238, but does not affect the level of total Histone H4 protein, detected by Western analysis. OD450nm readings are shown in the top figure, while the corresponding Western blot using the Acetylated Lysine mouse mAb (Ac-K-103) #9681 (left panel) or Histone H4 Antibody #2592 (right panel), is shown in the bottom figure.

Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated COS cells and kit assay optical density readings. COS cells were treated with TSA (4 µM overnight). An acid extraction was performed for cell lysis in the presence of 5 mM sodium butyrate.

PathScan® Acetyl-Histone H4 Sandwich ELISA Kit #7238

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Figure 1: Treatment of Jurkat cells with TSA causes accumulation of acetylation on Histone H4, detected by Sandwich ELISA Kit #7238, but does not affect the level of total Histone H4 protein, detected by Western analysis. OD450nm readings are shown in the top figure, while the corresponding Western blot using the Acetylated Lysine mouse mAb (Ac-K-103) #9681 (left panel) or Histone H4 Antibody #2592 (right panel), is shown in the bottom figure.

Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated COS cells and kit assay optical density readings. COS cells were treated with TSA (4 µM overnight). An acid extraction was performed for cell lysis in the presence of 5 mM sodium butyrate.

Product Description

The PathScan® Acetyl-Histone H4 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on histone H4. A Histone H4 antibody has been coated onto the microwells. After incubation with cell lysates, Histone H4 is captured by the coated antibody. Following extensive washing, an Acetylated-Lysine Rabbit mAb is added to detect the acetylated lysines on the Histone H4 protein. Anti-Rabbit IgG, HRP-Linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of acetylated Histone H4.

Antibodies in kit are custom formulations specific to kit.

Protocol

ELISA Colorimetric (Lyophilized)

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

Microwell strips: Bring all to room temperature before use.

Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.

HRP-Linked Antibody*: Supplied lyophilized as a red colored cake or powder. Add 1.0 ml of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.

Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

C. Test Procedure

After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4°C immediately.

Cell lysates can be undiluted or diluted with Sample Diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical kit assay results across a range of lysate concentration points.

Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.

Gently remove the tape and wash wells:

Discard plate contents into a receptacle.

Wash 4 times with 1X Wash Buffer, 200 µl each time for each well.

For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.

Clean the underside of all wells with a lint-free tissue.

Add 100 µl of reconstituted Detection Antibody (green color) to each well (refer to Section A, Step 2). Seal with tape and incubate the plate at 37°C for 1 hr.

Repeat wash procedure (Section C, Step 4).

Add 100 µl of reconstituted HRP-Linked secondary antibody (red color) to each well (refer to Section A, Step 3). Seal with tape and incubate the plate for 30 min at 37°C.

Repeat wash procedure (Section C, Step 4).

Add 100 µl of TMB Substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.

Add 100 µl of STOP Solution to each well. Shake gently for a few seconds.

NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H4 Sandwich ELISA Kit detects endogenous levels of acetylated histone H4. Using this Sandwich ELISA Kit #7238, acetylated lysines on Histone H4 are detected when treated with TSA in Jurkat cells. However, the levels of Histone H4 remain unchanged, as shown by Western analysis using the Histone H4 Antibody #2592 (figure 1). COS and NIH 3T3cells treated with TSA show similar results (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human, Mouse, Monkey

Background

Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).